PUB 301 Lecture 8 PDF

PUB 301 Lecture 8 Sterilization: Destruction or removal of all forms of microbial life including endospores (exception: prions) Commercial Sterilization: Sufficient heat to kill endospores of Clostridium botulinum in canned food Disinfection: removal/destruction of vegetative pathogens but not e...

PUB 301 Lecture 8 Sterilization: Destruction or removal of all forms of microbial life including endospores (exception: prions) Commercial Sterilization: Sufficient heat to kill endospores of Clostridium botulinum in canned food Disinfection: removal/destruction of vegetative pathogens but not endospores/resting stages Bacterial populations die at a constant logarithmic rate Effectiveness of Antimicrobial Treatment Depends on: Number of microbes Environment (organic matter, temperature, biofilms) Time of exposure Microbial characteristics Actions of Antimicrobial Agents Alteration of membrane permeability Damage to proteins Damage to nucleic acids Physical Methods of Microbial Control Heat: Kills microorganisms by denaturing enzymes, changing the structure of these proteins Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min Thermal death time (TDT): Time to kill all cells in a culture Moist Heat Sterilization Kills by denaturing proteins – breaking H bonds (like an egg white frying) Boiling kills vegetative forms of bacterial pathogens, almost all viruses and fungi/fungal spores in about 10 min Reliable sterilization requires temperatures above the boiling point of water Exceptions: Some hepatitis viruses can survive boiling for up to 30 min. Some bacterial endospores can survive 20+ hours  Sterilization in an autoclave is most effective when organisms are directly in contact with the steam or in an aqueous solution Under these conditions steam and pressure of 15psi (121°C) will kill all organisms and their endospores in ~15min Atmospheric Pressure and Altitude Atmospheric pressure drops at higher altitudes Therefore the pressure on autoclaves need to be set higher than at sea level Autoclave - Moist heat denatures proteins - Like a pressure cooker Steam Sterilization Steam must contact item’s surface Physical Methods of Microbial Control: Heat Pasteurization reduces spoilage organisms and pathogens 63°C for 30 min for milk High temperature, short time (HTST): 72°C for 15 sec Sterilization (refrigeration not required) Ultra high temperature (UHT): 140°C for 1% v/v Phenolics: Lysol Bisphenols: Hexachlorophene, Triclosan Disrupt plasma membranes Remain active in organic matter Stable and persist for long periods of time Use for pus, saliva & feces Effective against Gram positive Staphylococci and Streptococci bacteria and mycobacteria Types of Disinfectants: Biguanides Biguanides: Chlorhexidine Disrupt plasma membranes Combined with detergents and alcohol in surgical hand scrubs Low toxicity to skin Biocidal to most bacteria and fungi Not sporicidal Not effective against mycobacteria, endospores and protozoan cysts Types of Disinfectants: Halogens Iodine Exact mode of action unknown, probably combines with amino acids of enzymes and cellular proteins Effective against all types of bacteria, many endospores, various fungi and some viruses Available: in aqueous alcohol solution as tincture or as an iodophor in combination with an organic molecule from which iodine is released slowly e.g. Betadine Used mainly for skin disinfection and wound treatment Iodine tablets for treating camping water Types of Disinfectants: Halogens Chlorine Exact mode of action unknown, strong oxidizing agent that prevents cellular enzyme system from functioning Used as a gas or in water as hypochlorous acid (HOCl) Liquid form of compressed gas used for disinfecting drinking water and swimming pools Chloride compounds, sodium hypochlorite (NaOCl) bleach e.g. Chlorox used as household disinfectant Chloramines contain both chlorine and ammonia Glassware and eating utensil sanitizer Municipal drinking water treatment Toxic to fish Types of Disinfectants: Alcohols Ethanol, isopropanol Denatures proteins, dissolves lipids, disrupts membranes Acts before evaporating leaving no residue Kill bacteria and fungi but not non-enveloped viruses or endospores Not effective in wounds, causes coagulation of protein under which bacteria grow Types of Disinfectants: Heavy metals Silver, mercury, copper and zinc Oligodynamic action by small amounts Denatures proteins Silver with other anti-microbial drugs effective for burns, wounds and catheters Mercury more toxic and used for mildew control in paints Copper sulphate used as algaecide's in reservoirs, ponds and swimming pools Zinc chloride common ingredient in mouthwashes Zinc oxide most widely used anti- fungal Types of Disinfectants: Surface-active agents or surfactants Soap & water Not antiseptic Emulsifier Cleans by washing away oil and dead cells Acid-anionic (negatively charged) detergents Reacts with plasma membranes Sanitizer for dairy utensils and equipment Wide spectrum includes thermoduric bacteria Non-toxic, non-corrosive and fast acting Types of Disinfectants: Quaternary Ammonium Compounds Positively charged cationic detergents Denature proteins, disrupt plasma membrane Bactericidal especially Gram positives Fungicidal, amoebicidal and virucidal against enveloped viruses Does not kill endospores or mycobacteria Certain Gram negatives can survive and grow actively Zephiran: Mr. Clean Cepacol cetyl pyridimium chloride Types of Disinfectants: Aldehydes Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, –SH) Used by morticians in embalming Formaldehyde Gas is excellent disinfectant More commonly available as formalin (37% aqueous solution) Glutaraldehyde Used to disinfect hospital instruments 2% solution (Cidex) tuberculocidal, virucidal after contact for 10 min sporicidal after 3 – 10 hr Types of Disinfectants: Gaseous sterilants Chemicals that sterilize in closed chamber Ethylene oxide Denature proteins Kills all microbes and endospores Lengthy exposure period of 4 –18 hrs Toxic and explosive so mixed with non-flammable gas such as CO2 or nitrogen Used for sterilizing hospital supplies and equipment Types of Disinfectants: Peroxygens (Oxidizing Agents) Ozone (O3) Highly reactive form of oxygen Generated by passing oxygen through high voltage electrical discharges Helps neutralize taste and odour, used with chlorine in water treatment Types of Disinfectants: Peroxygens (Oxidizing Agents) Hydrogen peroxide (H2O2) Common antiseptic in households and hospitals Not suitable for open wounds as it slows down healing Quickly broken down by catalase present in human cells Effective on inanimate objects and surfaces especially against aerobes and facultative anaerobes Increasing use in food industry to disinfect packaging material by passing through hot solution of hydrogen peroxide Contact lens disinfection Types of Disinfectants: Peroxygens (Oxidizing Agents) Benzoyl peroxide Over-the-counter medication for acne Useful for treating wounds infected with anaerobes Per-acetic acid One of the most effective liquid chemicals used as sporicides Short disinfection times 5 – 30 mins Used for food-processing and medical equipment as it leaves no toxic residues and is minimally affected by organic matter Microbial Characteristics and Microbial Control Factors that prevent chemical disinfection: Lipopolysaccharide layer and porins of Gram negative bacteria Lipid-rich cell wall of Mycobacteria Bacterial endospores Fungal spores Protozoan (oo)cysts Non-enveloped viruses Prions Types of Disinfectants: Chemical food preservatives, Organic acids & salts Interferes with metabolism & plasma membranes Sorbic acid or potassium sorbate, benzoic acid or sodium benzoate, and calcium propionate Control molds and bacteria in foods e.g. cheese, soft drinks Considered safe in foods Sodium nitrate and nitrite Prevents endospore germination Preserves red meat colour by reacting with blood components Added to processed meat products e.g. ham, bacon, hot dogs & sausages React with amino acids to form carcinogenic nitrosamines Types of Disinfectants: Chemical food preservatives - Antibiotics Nisin which is tasteless, easily digested and non-toxic prevents growth of endospore- forming spoilage bacteria Natamycin anti-fungal & antibiotic approved for use in foods e.g. cheese Antibiotics 1928: Fleming discovered penicillin, produced by a mould Penicillium notatum 1940: Howard Florey and Ernst Chain performed first clinical trials of penicillin Sources of antibiotics come from other microorganisms Antibiotic Sources Streptomyces Filamentous bacteria found in soil Most antibiotic producing microbes have some kind of sporulation process Actinomycetes Cyanobacteria Spectrum of Anti-microbial Activity Narrow spectrum of microbial activity: drugs that affect a narrow range of microbial types Broad-spectrum antibiotics: affect a broad range of gram-positive or gram-negative bacteria The Action of Antibiotics Bactericidal Kill microbes directly Bacteriostatic Prevent microbes from growing Does not affect human cells Broad-spectrum antibiotics can destroy normal flora in gut and other parts of the body Yeast overgrowth can result Antibiotic resistance can also develop The Action of Antimicrobial Drugs Antibiotics: Inhibitors of Cell Wall Synthesis Penicillin prevents bacterial cell wall peptidoglycan synthesis Penicillin ß-lactam ring Highly reactive ring interferes with bacterial cell wall synthesis Natural penicillins Extracted from Penicillium cultures Penicillin G (injected) and Penicillin V (oral) Narrow spectrum of activity Susceptible to penicillinases (β-lactamases) Semisynthetic penicillins Contain chemically added side chains, making them resistant to penicillinases Antibacterial Antibiotics: Inhibitors of Cell Wall Synthesis Penicillinase-resistant penicillins Methicillin and oxacillin Extended-spectrum penicillins Effective against gram-negatives as well as gram-positives Aminopenicillins: ampicillin, amoxicillin Penicillins plus β-lactamase inhibitors Contain clavulanic acid, a noncompetitive inhibitor of penicillinase Antibacterial Antibiotics: Inhibitors of Cell Wall Synthesis Carbapenems Substitute a C for an S and add a double bond to the penicillin nucleus Broad spectrum Primaxin, doripenem Monobactam Synthetic; single ring instead of the β-lactam double ring Low toxicity; works against only certain gram- negatives Aztreonam Antibacterial Antibiotics: Inhibitors of Cell Wall Synthesis Cephalosporins Work similar to penicillins β-lactam ring differs from penicillin Grouped according to their generation of development Polypeptide antibiotics Bacitracin Topical application; works against gram-positives Vancomycin Glycopeptide Last line against antibiotic-resistant MRSA Cephalosporins Cephalosporins 2nd, 3rd, and 4th generations more effective against gram-negatives Resistant to penicillinase Cefotaxime, cefixime The Action of Antimicrobial Drugs Antibiotics: Inhibitors of Protein Synthesis Chloramphenicol Binds 50S subunit, inhibits peptide bond formation Adverse side effects e.g. suppresses formation of blood cells leading to aplastic anemia Broad spectrum Aminoglycosides Changes shape of 30S subunit Streptomycin, neomycin, gentamicin Broad spectrum and effective against Pseudomonas infections in cystic fibrosis Antibiotics: Inhibitors of Protein Synthesis Tetracyclines Binds 30S subunit Broad spectrum Also effective against intracellular bacteria Absorbed by metal ions e.g. Ca Discolours teeth in children Liver damage in pregnant women Antibiotics: Inhibitors of Protein Synthesis Streptogramins Binds 50S subunit, inhibits translation Cyclic peptides Gram-positives pristinamycin Antibiotics: Inhibitors of Protein Synthesis Macrolides Gram-positives Binds 50S, prevents translocation Large macrolide ring Antibiotics: Inhibitors of Protein Synthesis Oxazolidinones New class of FDA approved antibiotics in 2001 (after 25 years) Vancomycin resistant bacteria Linezolid Gram-positives Binds 50S subunit, prevents formation of 70S ribosome Antibiotics: Injury to the Plasma Membrane Polymyxin B Injures plasma membranes Effective against gram negative bacteria such as Pseudomonas Topical Combined with bacitracin and neomycin in over-the-counter preparation Antibiotics: Inhibitors of Nucleic Acid Synthesis Rifamycin Inhibits mRNA synthesis Able to penetrate tissues and reach therapeutic levels in CSF and abscesses Effective in treating tuberculosis caused by intracellular Mycobacteria located in macrophages Antibiotics: Inhibitors of Nucleic Acid Synthesis Quinolones and fluoroquinolones Inhibits DNA gyrase needed for DNA replication Nalidixic acid effective in treatment of urinary tract infections Ciprofloxacin, second generation fluoroquinolone, is broader spectrum used in treatment of anthrax Antibiotics: Competitive Inhibitors Sulphonamides (sulphur drugs) Structural similarity to para-aminobenzoic acid (PABA) a folic acid precursor Inhibits folic acid synthesis Sulphamethoxazole with Trimethoprim Broad spectrum especially used for treating Pneumocystis pneumonia an opportunistic infection in AIDS Disk-Diffusion Test a.k.a Kirby-Bauer test Filter disks soaked with known concentrations of antibiotics Larger zone of inhibition indicates greater sensitivity Bacteria reported as sensitive, intermediate or resistant E Test: Advanced Diffusion Method to Determine the Minimal Inhibitory Concentration Plastic coated strips contain gradient of antibiotic concentrations Inhibitory Concentrations of Antibiotics MIC: Minimal inhibitory concentration The lowest antibiotic concentration that prevents visible bacterial growth Does not distinguish between bacteriostatic or bactericidal *Remember: bacteriostatic: agent that prevents bacteria from reproducing Bactericidal: substance that kills bacteria Inhibitory Concentrations of Antibiotics MBC: Minimal bactericidal concentration MLC minimal lethal concentration Determined using broth dilution tests Antibiotic Resistance Misuse of antibiotics that selects for resistance mutants includes: Using outdated or weakened antibiotics Using antibiotics for the common cold and other inappropriate conditions Use of antibiotics in animal feed Failure to complete the prescribed regimen Using someone else's leftover prescription Some facts Penicillin was over the counter until the 1950s Many antibiotics are still sold over the counter in much of the developing world US – 50% of documented antibiotic treatment is unnecessary Factory farmed animals given antibiotics daily (in US this accounts for 80% of antibiotics used yearly) Apple, Pears, citrus farming – bacterial infections Bacterial generation times of 20 min Compared to 10 years that it generally takes to develop and ensure safety of new antibiotics Deaths by 2050, WHO Mechanisms of Antibiotic Resistance 1. Enzymatic destruction of drug Beta-lactamases are bacterial penicillinases that cleave beta-lactam ring of the penicillin molecule MRSA (methicillin-resistant Staphylococcus aureus) Methicillin 1st semi-synthetic penicillin VRE (vancomycin resistant Enterococcus) Mechanisms of Antibiotic Resistance 2. Prevention of penetration of drug Gram negative mutants alter porin openings 3. Alteration of drug's target site Modification of protein synthesis sites Macrolide & tetracycline resistance 4. Rapid ejection of the drug Rapid efflux pumps out antibiotics Tetracycline resistance Consequences… What else do we lose? Surgery Artificial body replacements Transplants Dialysis Pneumonia 3 in 10 children Strep, skin infections (amputation) UK estimates 700,000 deaths a year can already be attributed in some way to antibiotic resistance 2050 projection 10,000,000 a year "The thoughtless person playing with penicillin treatment is morally responsible for the death of the man who succumbs to infection with the penicillin-resistant organism.“ Alexander Fleming Antibiotic Resistance Globally and in the News! https://www.theguardian.com/global-development/2018/feb/22/drug-resistant- superbug-deadly-typhoid-outbreak-pakistan http://www.cbc.ca/radio/day6/episode-321-women-s-march-on- washington-hamilton-s-one-last-time-jerry-maguire-circus-farewells- and-more-1.3941806/an-american-woman-just-died-from-a-superbug- resistant-to-26-different-antibiotics-1.3941881 Effects of Drug Combinations Synergism occurs when the effect of two drugs together is greater than the effect of either alone Amoxicillin a broader spectrum semi-synthetic penicillin Clavulanic acid a beta-lactamase inhibitor Antagonism occurs when the effect of two drugs together is less than the effect of either alone Penicillin and tetracycline Effects of Drug Combinations Future of Chemotheraputic Agents Antimicrobial Peptides immune system products in many organisms 100s identified (defensins) Disrupt microbial membranes that are rich in phospholipids Antisense Agents bind to DNA sites that cause pathogenic effects Phage Therapy peptides produced by bacteriophages could be used as antimicrobials https://www.youtube.com/watch?v=zENv5EDElgA